Wnt/b-catenin signaling is a conserved developmental pathway that plays important roles in human disease. Mutations in adenomatous polyposis coli, a Wnt pathway component, are responsible for familial adenomatous polyposis syndrome and 80% of nonhereditary colorectal cancers. Since its identification two decades ago, however, the function of APC in Wnt signaling remains poorly understood. APC is part of a multi-protein complex that promotes ubiquitin-mediated degradation of the transcriptional coactivator, beta-catenin. Loss of APC function (due to truncating mutations or downregulating APC levels by RNAi) results in elevated b-catenin levels and ligand-independent activation of the Wnt pathway. We have developed a monoclonal antibody against LRP6, the Wnt coreceptor that inhibits Wnt3a-mediated activation of the Wnt pathway in cultured mammalian cells. Current models of Wnt signaling suggest that APC functions exclusively downstream of Wnt receptors. Surprisingly, our anti-LRP6 antibody (as well as LRP6 RNAi constructs) inhibits Wnt signaling in several cancer cell lines with mutation of APC as well as in cells depleted of APC by siRNA. Treatment of APC mutant cancer cells with the anti-LRP6 antibody downregulates intracellular levels of beta-catenin, consistent with effects on b-catenin degradation. In this proposal, we seek to uncover the link between APC and LRP6 in regulating Wnt pathway activation. We will assess whether loss of LRP6 function by anti-LRP6 antibody treatment or RNAi inhibits Wnt signaling in a larger panel of cancer lines with mutations in APC. We will test the possibility tha the Wnt pathway is activated at the level of the Wnt coreceptors (Frizzled and LRP6) upon loss of APC function. We will test whether other proteins upstream of the beta-catenin degradation complex are required for activation of the Wnt pathway upon APC loss of function by RNAi knockdown or expression of dominant-negative proteins. We predict that APC and LRP6 compete for binding to the beta-catenin degradation complex, and we will test this hypothesis in cultured cells, Xenopus egg extract, and with purified proteins. Finally, we propose to provide in vivo evidence using Xenopus embryos to confirm that the activation of Wnt target gene transcription in APC- morphant embryos can be blocked by LRP6 downregulation. These studies have the potential to provide insight into the function of an important tumor suppressor, APC, and to directly impact the development of therapeutics for the treatment of Wnt-driven diseases due to mutations in APC.

Public Health Relevance

This proposal focuses on a new mechanism of action for the adenomatous polyposis coli protein (APC) in the Wnt signal transduction pathway. We find that activation of the Wnt pathway due to APC mutation or downregulation is dependent on the Wnt co-receptor, LRP6. We propose to perform biochemistry and Xenopus embryo studies to test our model that APC and LRP6 interact to regulate Wnt signaling.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Intercellular Interactions Study Section (ICI)
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Dunsmore, Sarah
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Vanderbilt University Medical Center
Anatomy/Cell Biology
Schools of Medicine
United States
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Saito-Diaz, Kenyi; Benchabane, Hassina; Tiwari, Ajit et al. (2018) APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway. Dev Cell 44:566-581.e8
Ng, Victoria H; Hang, Brian I; Sawyer, Leah M et al. (2018) Phosphorylation of XIAP at threonine 180 controls its activity in Wnt signaling. J Cell Sci 131:
Li, Bin; Orton, Darren; Neitzel, Leif R et al. (2017) Differential abundance of CK1? provides selectivity for pharmacological CK1? activators to target WNT-dependent tumors. Sci Signal 10:
Rodriguez-Blanco, J; Pednekar, L; Penas, C et al. (2017) Inhibition of WNT signaling attenuates self-renewal of SHH-subgroup medulloblastoma. Oncogene 36:6306-6314
Ma, Yufang; Wang, Lihong; Neitzel, Leif R et al. (2017) The MAPK Pathway Regulates Intrinsic Resistance to BET Inhibitors in Colorectal Cancer. Clin Cancer Res 23:2027-2037
Lebensohn, Andres M; Dubey, Ramin; Neitzel, Leif R et al. (2016) Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling. Elife 5:
Broadus, Matthew R; Chen, Tony W; Neitzel, Leif R et al. (2016) Identification of a Paralog-Specific Notch1 Intracellular Domain Degron. Cell Rep 15:1920-9
Astudillo, Luisana; Da Silva, Thiago G; Wang, Zhiqiang et al. (2016) The Small Molecule IMR-1 Inhibits the Notch Transcriptional Activation Complex to Suppress Tumorigenesis. Cancer Res 76:3593-603
Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan et al. (2016) Wnt pathway activation by ADP-ribosylation. Nat Commun 7:11430
Wang, Zhenghan; Tacchelly-Benites, Ofelia; Yang, Eungi et al. (2016) Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase. Genetics 203:269-81

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